The present invention relates generally to the field of molecular genetics. More particularly, the invention relates to a polynucleotide sequence encoding a variant xcex16 subunit of the human GABAA neurotransmitter receptor, the sequence variant predicting sensitivity to both benzodiazepine drugs and ethanol.
Human heritability studies using twins and adoptees have indicated that alcoholism is a complex disorder having a genetic component. (Hesselbrock, xe2x80x9cThe Genetic Epidemiology of Alcoholismxe2x80x9d in The Genetics of Alcoholism, Edited by Begleiter H, Kissin B. New York, Oxford University Press, pp 17-39 (1995)). Sons of alcoholics (SOAs) are a group at high risk for developing alcoholism (Cloninger et al., Arch Gen Psychiatry 38:861 (1981)), and so have been the focus of numerous studies on the subjective, psychomotor, physiological and biochemical responses to ethanol. (Schuckit, Alcohol Clin Exp Res 12:465 (1988); Newlin et al., Psychol Bull 108:383 (1990); Pollock, Am J Psychiatry 149:1534 (1992)). These studies have identified several differences between SOAs and male control subjects which can provide clues to the basis of increased risk of developing alcoholism.
One of the distinctions between SOAs and male control subjects relates to differential sensitivity to benzodiazepine drugs (BZD) and ethanol. More particularly, SOAs have been shown to be significantly less sensitive to BZD (Ciraulo et al., Am J Psychiatry 146:1333 (1989); Cowley et al., Alcohol Clin Exp Res 16:1057 (1992); Cowley et al., Alcohol Clin Exp Res 18:324 (1994)) and ethanol (Schuckit et al., Arch Gen Psychiatry 53:202 (1996)) when compared with male control subjects. The genetic and neurobiological mechanisms underlying this diminished sensitivity is unclear, because these drugs affect multiple neurotransmitter systems in the central nervous system. (CNS) (Deitrich et al., Pharmacol Rev 41:489 (1989)).
xcex3-Aminobutyric acid (GABA) is a key inhibitory neurotransmitter in the mammalian CNS. GABA subtype A (GABAA) receptors are chloride channels that specifically bind benzodiazepine drugs with high affinity (Luddens et al., Neuropharmacology 34:245 (1995)) to result in chloride ion influx. Molecular analysis has revealed that GABAA receptor channels are heterooligomeric structures composed of several distinct polypeptide subunits (xcex11-6, xcex21-3, xcex31-3, and xcex4). (Burt et al., FASEB J 5:2916 (1991)).
Two lines of evidence have implicated the GABAA receptor in differential sensitivity to alcohol. First, cerebellar membranes of xe2x80x9cANTxe2x80x9d and xe2x80x9cATxe2x80x9d rats exhibited differential affinity for BZD. (Uusi-Oukari et al., J Neurochem 54:1980 (1990)). The Alcohol-sensitive ANT (Alcohol Non-Tolerant) and alcohol-insensitive AT (Alcohol Tolerant) lines of rats have been selectively bred to exhibit differences in sensitivity to ethanol-induced motor impairment. An amino acid substitution Arg100Glu in the GABAA xcex16 receptor is believed to be at least partially responsible for the difference in alcohol sensitivity which characterizes these two rat lines. The alcohol insensitive AT line carries the Arg100 form of the receptor and is diazepam insensitive when compared with the Glu100 GABAA xcex16 receptor. (Korpi et al., Nature 361:356 (1993)).
In a second line of evidence, the GABAA xcex32 subunit was implicated in the differential sensitivity of long-sleep (LS) and short-sleep (SS) mouse lines to acutely administered alcohol. (Wafford et al., Science 249:291 (1990)). An in vitro mutagenesis and expression system employing Xenopus oocytes was used to demonstrate that alternative RNA splicing of a region of the GABAA xcex32L subunit, which encodes a consensus protein kinase C (PKC) phosphorylation site, was critical for modulation by ethanol. (Wafford et al., Neuron 7:27 (1991); Wafford et al., FEBS Lett 313:113 (1992)). Clearly there remains a need to better understand the genetic basis for differential sensitivity to benzodiazepine drugs and alcohol in humans.
Compositions and methods based on a polymorphism in the gene encoding the xcex16 subunit of the human GABAA neurotransmitter receptor are disclosed. This polymorphism results in the substitution of a serine residue for a proline residue ordinarily present at amino acid position 385 of the GABAA xcex16 polypeptide sequence. In a first set of experiments, we demonstrate that patients having the Pro385Ser polymorphism exhibit a reduced sensitivity to diazepam, a benzodiazepine drug known in the art to mimic a subject""s response to ethanol, when compared with patients having the proline residue at amino acid position 385. The Pro385Ser polymorphism was found to be associated with less change in smooth pursuit eye movement gain after intravenous diazepam was administered to children of alcoholics (COAs). In a second set of experiments, we demonstrate that patients having the Pro385Ser polymorphism exhibit a reduced sensitivity to ethanol when compared with patients having the proline residue at amino acid position 385. Thus, the polymorphism was associated with decreased sensitivity to ethanol and benzodiazepine drugs.
Herein, we have discovered two genetic differences or xe2x80x9cpolymorphismsxe2x80x9d that occur in the gene sequence encoding the xcex16 subunit of the human GABAA neurotransmitter receptor. In the most prevalent form of the xcex16 subunit of the human GABAA neurotransmitter receptor, a proline residue is present at amino acid position 385 given by the sequence provided by Hadingham et al., Mol Pharmacol 49(2):253-259 (1996), herein incorporated by reference. This form of the xcex16 receptor subunit protein or the xcex16 receptor subunit protein-encoding polynucleotide having a proline residue at amino acid position 385 is referred to throughout this disclosure as xe2x80x9cPro385xe2x80x9d.
In this invention, we have discovered a first polymorphism, called xe2x80x9cPro385Serxe2x80x9d or xe2x80x9cSer385xe2x80x9d, in the xcex16 subunit of the human GABAA neurotransmitter receptor. This form of the xcex16 subunit of the human GABAA neurotransmitter receptor is characterized by a substitution of a serine residue for the proline residue which is ordinarily present at amino acid position 385. In some contexts, the term xe2x80x9cPro385Serxe2x80x9d or xe2x80x9cSer385xe2x80x9d refers to a polymorphism in a polynucleotide encoding xcex16 protein (in which case the polymorphism is with reference to codon 385 of the xcex16-encoding polynucleotide), or to the xcex16 protein itself (in which case the polymorphism is with reference to amino acid position 385 of the xcex16 polypeptide sequence given by Hadingham et al., Mol Pharmacol 49(2):253-259 (1996). In other contexts, the term Pro385Ser or Ser385 refers to a polymorphism in a polynucleotide encoding a fragment of xcex16 protein (in which case the polymorphism is with reference to codon 385 of the xcex16 fragment-encoding polynucleotide), or to a fragment of the xcex16 protein itself (in which case the polymorphism is with reference to amino acid position 385 of the xcex16 polypeptide sequence given by Hadingham et al., Mol Pharmacol 49(2):253-259 (1996). The Pro385Ser polymorphism can also be referred to as GABRA6 1236C greater than T to indicate the single nucleotide change at position 1236 which confers the substitution of the serine amino acid residue for the proline amino acid residue at amino acid position 385.
We have also discovered a second polymorphism, called G1031C, but this nucleotide change did not alter the amino acid sequence of the xcex16 receptor. The G1031C polymorphism can also be referred to as GABRA6 1031G greater than C.
The Pro385Ser polymorphism occurs within a portion of the xcex16 subunit of the human GABAA neurotransmitter receptor corresponding to the second intracellular domain of the receptor near a putative protein kinase C phosphorylation site. By the phrase, xe2x80x9ca portion of the xcex16 subunit of the human GABAA neurotransmitter receptorxe2x80x9d is meant a segment of the xcex16 polypeptide sequence that includes at least 50-100 amino acids, more preferably at least 20 contiguous amino acids, and even more preferably at least 3, 6 or 10 contiguous amino acids. The Pro385Ser polymorphism exhibits a rarer-allele frequency of 0.08, whereas, the G1031C polymorphism exhibits a rarer-allele frequency of 0.47. By xe2x80x9cAllelexe2x80x9d or xe2x80x9callelic variantxe2x80x9d is meant the natural polynucleotide sequences corresponding to polymorphisms present in human beings.
In the following disclosure, we first demonstrate that patients having the Pro385Ser polymorphism exhibit a reduced sensitivity to diazepam, a benzodiazepine drug known in the art to mimic a subject""s response to ethanol, when compared with patients having the proline residue at amino acid position 385. The Pro385Ser polymorphism was found to be associated with less change in smooth pursuit eye movement gain after intravenous diazepam was administered to children of alcoholics (COAs). In contrast, the G1031C polymorphism did not correlate with diazepam sensitivity. Second, we demonstrate that patients having the Pro385Ser polymorphism exhibit a reduced sensitivity to ethanol when compared with patients having the proline residue at amino acid position 385.
Biological tools, therapeutics, and methods of use of the foregoing are provided. Further, embodiments that employ diagnostics or diagnostic kits, which are particularly useful for the rapid identification of the Pro385Ser polymorphism and, thus the determination of the relative sensitivity to a BZD drug or ethanol in an individual having a particular genotype, are provided. In the section below, we discuss the discovery of the Pro385Ser polymorphism in greater detail.
Genetic Polymorphisms in the GABAA Receptor xcex16 Subunit
Those having ordinary skill in the art will appreciate that differential sensitivity to benzodiazepine drugs is a useful indicator of brain differences correlating with behavioral variation, including susceptibility to alcoholism. In rats, for example, variation in alcohol and benzodiazepine sensitivity has been correlated with an inherited variant of the GABAA xcex16 receptor. Significantly, responses to eye movement tasks which can be measured as saccade gain, peak saccade velocity, and smooth pursuit gain, have been demonstrated to be reliable and reproducible within and between testing sessions, and to be objective, quantifiable measures affected by BZD in a dose-dependent manner. (Roy-Byrne et al., Psychopharmacology 110:85-91 (1993); Roy-Byrne et al., Biol Psychiatry 38:92 (1995)). Accordingly, in the procedures described below, eye movement testing provided a convenient means of monitoring BZD and ethanol sensitivity in human subjects.
Based on the finding that SOAs exhibited decreased sensitivity to BZD, and the fact that rodent genetic studies implicated GABAA receptors in response to BZD and alcoholism, we first investigated whether polymorphisms in the GABAA xcex16 subunit gene were associated with differences in BZD sensitivity in children of alcoholics. The procedures employed during the development of the invention involved correlating BZD sensitivity and genotype. More particularly, the procedures involved assessing sensitivity to diazepam in COAs using two eye movement tasks: peak saccadic velocity and average smooth pursuit gain. Genetic variation within the GABAA xcex16 receptor gene coding region was evaluated in 56 unrelated COAs by the single strand conformational polymorphism method. Association analysis using a Ser385 substitution, the synonymous variant G1031C and GABAA xcex16 haplotype was performed with smooth pursuit gain and peak saccadic velocity as dependent variables. As indicated below, the Ser385 genotype correlated with less diazepam-induced impairment in average smooth pursuit gain (t=1.954, df=53, p=0.04), but not peak saccadic eye velocity. The synonomous G1031C polymorphism was not associated with altered diazepam effects on either eye movement task.
Subjects in the studies described herein were 26 unrelated sons and 30 daughters of alcoholic fathers. All subjects were Caucasian. Subjects ranged from 18-25 years of age. Psychiatric disorders were diagnosed by DSM-III-R criteria and using the Structured Clinical Interview for DSM-III-R (SCID). (Spitzer et al., Structured Clinical Interview for DSM-III-R Patients Edition (SCID-P, 9/1/89 version), New York, Biometrics Research Department New York Psychiatric Institute, (1989)). The proband and at least one first-degree relative were interviewed with the Family Informant Schedule and Criteria. (Mannuzza et al., Family Informant Schedule and Criteria (FISC) Anxiety Disorders Clinic, New York, New York Psychiatric Institute, 1985), an extended version of the Family History-Research Diagnostic Criteria (Andreasen et al., Arch Gen Psychiatry 43:421 (1986)) and with the Family History Assessment Module from the SSAGA, a structured diagnostic interview used in the Collaborative Study on the Genetics of Alcoholism. (Bucholz et al., J Stud Alcohol 55:149 (1994)).
The modalities described above enabled us to confirm the diagnosis of alcohol dependence in the father, how many other first- and second-degree relatives were affected, and to establish family history of other psychiatric disorders. COAs were free of lifetime DSM-III-R Axis I or Axis II psychiatric or substance use disorders, except adjustment disorder and had no history of antisocial personality disorder. All COAs were medically healthy and by their report had taken no medication for at least one month nor had they used a benzodiazepine more than once. All subjects had negative urine drug screens, normal xcex3-glutamyltransferase, and normal mean corpuscular volume. Example 1 describes the methods that were used to measure BZD sensitivity in human subjects in greater detail. In the section below, we describe the association of diazepam sensitivity with the Pro385 and Ser385 polymorphisms.
Diazepam Sensitivity and the Pro385 or Ser385 GABAA xcex16 Genotype
Initially, the saccadic eye movement velocity and smooth pursuit eye movement gain was measured in the 56 COAs tested. Two baseline measurements for each variable were taken and averaged. The differences from this baseline were then evaluated at set time points after each of four diazepam doses. The data points were used to determine, by a trapezoidal technique, the integrated area under the resulting curve. The area under the curve for the dependent variable was then divided by the area under the curve for plasma diazepam levels to correct for individual variability in diazepam levels. The ratios of the areas under the curves for COAs with the Ser385 homozygous genotype and the Pro/Ser heterozygous genotype were then compared by t-test. There were no Ser/Ser homozygotes in the test population of COAs. For the G1031C genotype, a one-way ANOVA was used for assessing association.
The genotypes at each locus were also tested for Hardy-Weinberg equilibrium by Fisher""s Exact Test. A maximum likelihood method was used to estimate haplotype frequencies in the double heterozygotes. (Hill, Heredity 33:229 (1974); Weir, Genetic data analysis II, Sunderland, Mass., Sinauer Associates (1996)). Haplotype frequencies from single heterozygotes were determined by direct counting. Normalized linkage disequilibrium (xcex94) and linkage disequilibrium (D) were calculated using these haplotype frequencies.
The polymorphic variation in the GABAA xcex16 coding region from the 56 unrelated COAs was then confirmed using SSCP followed by DNA sequencing. (See Example 2). The two relatively abundant polymorphisms that were identified in the xcex16 coding region are presented in Table 4 (Table 4 can be found in Example 2, infra). A thymidine to cytosine transition at nucleotide 1236 of the coding sequence was identified and the transition results in a substitution of a proline residue by a serine residue at codon 385. This polymorphism was designated xe2x80x9cPro385Serxe2x80x9d or xe2x80x9cGABRA6 1236C greater than T. The second polymorphism identified was a silent G to C substitution at nucleotide 1031, and was designated xe2x80x9cG1031Cxe2x80x9d or GABRA6 1031G greater than C. As presented in the appended sequence listing, the Pro385 sequence is given by SEQ ID NO: 11, and the Ser385 sequence is given by SEQ ID NO: 12. The G1031C GABAA xcex16 sequence is given by SEQ ID NO: 13 while the C1031C sequence is given by SEQ ID NO: 14.
PCR-RFLP assays were also used to facilitate rapid genotyping based on the two GABAA xcex16 polymorphisms. (See Example 2). All subjects genotyped by SSCP-analysis and PCR-RFLP yielded identical results. The frequencies of the rarer Pro385Ser and 1031C alleles in the COAs were 0.08 and 0.47, respectively. Both genotype distributions were consistent with expectations based on Hardy-Weinberg equilibrium estimates. The level of linkage disequilibrium between Pro385Ser and G1031C was significant (xcex94=1.00, D=0.0401, "khgr"2=10.8, d.f.=1, p less than 0.0005).
An association between Ser385 genotype and average smooth pursuit eye movement gain following intravenous diazepam administration was observed (t=1.95, df=53, P=0.04). (See Table 1). COAs having the Pro385Ser allele exhibited a reduced sensitivity to diazepam relative to a group consisting of COAs having the Pro385 genotype. By reduced sensitivityxe2x80x9d is meant that a level of sensitivity that is lower than the level of sensitivity which characterizes individuals having the Pro385 genotype. The Pro385Ser allele, however, was not associated with the peak velocity of saccadic eye movement (t=0.171, df=52, p=0.865). Further, no association between the synonymous G1031C polymorphism and either of the two eye movement tasks was found. The numbers of subjects used in the two analyses differed because one individual was so sensitive to diazepam that data for saccadic velocity could not be collected at the highest diazepam dose.
Next, we looked to correlate polymorphic variants of the GABAA xcex16 receptor in COAs, specifically the Ser385 genotype, with saccadic eye movement velocity and smooth pursuit eye movement gain differences in COAs before and after diazepam administration, as disclosed in the section below.
The Pro385Ser Polymorphism and Benzodiazepine/Ethanol Insensitivity
Among the 13 subunits comprising the GABAA/BZD receptor complexes in mammals the xcex16 subunit is unique in its benzodiazepine agonist-insensitive pharmacology and in its restricted distribution. (Burt et al., FASEB J 5:2916 (1991)). GABAA xcex16 expression, for example, is limited to cerebellar granule cells. (Luddens et al., Neuropharmacology 34:245 (1995); Luddens et al., Nature 346:648 (1990)). In the genotyping procedure discussed above, we found that the level of response to alcohol in humans correlated with the GABAA xcex16 genotype. This discovery is the first indication that a difference in human diazepam response, and, thus, ethanol response can arise from a naturally occurring variant in a GABAA receptor subunit. Not wanting to be limited to any particular mechanism and offered only for the purposes of explanantion, we believe that diazepam binding to the xcex16 Ser385 receptor modulates GABAA currents, which results in the differences in smooth pursuit eye movement gain observed between subjects having the Pro385 genotype.
We did not observe an association between the Ser385 genotype and saccadic eye movement. Saccadic eye movements involve the parietal cortex, the frontal cortex and associated connections with the basal ganglia, as well as the superior colliculus and, finally, pre-motor areas in pons. (Henn et al., Rev Neurol 145:540 (1989)). Pursuit eye movements that were associated with Pro385Ser are predominantly mediated by multiple cortical areas and cerebellum. (Leigh et al., The Neurology of Eye Movement. 2 ed. Philadelphia, F. A. Davis Co., 1991). Hence, saccadic velocity and smooth pursuit gain provide a measure of BZD effects in brainstem and cortex/cerebellum, respectively. It is noteworthy that the association of Pro385Ser to pursuit eye movements but not to saccadic movements corresponds to the restricted expression of GABAA xcex16 to the cerebellum.
We also did not find any association between G1031C and diazepam sensitivity in spite of the fact that Pro385Ser and G1031C are in strong linkage disequilibrium. Although linkage disequilibrium between the two loci is strong, allele frequency differences between the two loci can account for lack of association with the more abundant G1031C synonymous variant.
Pro385 is conserved in the GABAA xcex16 receptors of rats and mice (Luddens et al., Nature 346:648 (1990)) (Kato, J Mol Biol 214:619 (1990)). The Ser385 amino acid residue is located in the second intracellular domain of the receptor and at a site ten amino acid residues removed from a consensus phosphorylation site for PKC. (Luddens et al., Nature 346:648 (1990)). Some believe that the domain can contribute to subtype specificity and intracellular regulatory mechanisms. (Olsen et al., FASEB J 4:1469 (1990)). Until the present disclosure, however, a function for this amino acid and domain has not been confirmed.
With respect to the GABAA xcex32L subunit, it is of interest that an alternatively spliced variant of xcex32 which contains an extra eight amino acids bears a consensus site for phosphorylation by PKC. (Whiting et al., Proc Natl Acad Sci USA 87:9966 (1990)). Site-directed mutagenesis of the alternatively spliced sequence indicates that the xcex32L subunit must be phosphorylated to confer ethanol sensitivity. (Wafford et al., Neuron 7:27 (1991); Wafford et al., FEBS Lett 313:113 (1992)). Not wanting to be limited to any particular mechanism and offered only for the purposes of explanation, we speculate that a similar role for this domain of the xcex16 subunit could enable the Pro385Ser variant to alter the sensitivity of the receptor to ethanol and BZD.
After determining that individuals with the Pro385Ser polymorphism exhibited a reduced sensitivity to benzodiazepine drugs when compared with patients having the Pro385 polymorphism, we verified that individuals with the Pro385Ser polymorphism also exhibited a reduced sensitivity to ethanol when compared with patients having the wild-type polymorphism. These results are described in the following section.
Individuals with the Pro385Ser Polymorphism Exhibit a Reduced Sensitivity to Ethanol
Alcohol abuse and dependence (alcoholism) are complex disorders that appear to reflect several genetic influences that together might explain 40% to 60% of the variance of risk. (Kendler et al., Arch Gen Psychiatry 54:178-184 (1997); Pickins et al., Arch Gen Psychiatry 48:19-28 (1991)). A low level of response (LR) to alcohol appears to be one route by which vulnerability is mediated. Even after controlling for other influences, LR is apparently both genetically influenced and associated with an enhanced risk for alcohol dependence. Schuckit and Smith, Arch Gen Psychiatry 53:202-210 (1996)). In humans, LR has been found to occur at a higher frequency among young drinking, but nonalcoholic, family history positive (FHP) men (Pollock Am J Psychiatry 149:1534-1538 (1991); Schuckit and Smith 1996; Newlin and Thomson Psychol Bull 108:383-402 (1990)), while identical twins are more similar on LR than fraternal twins. (Madden et al 1995; Rose et al 1994). Three follow-ups of subjects from alcohol challenges have revealed higher rates of alcoholism, alcohol-intake, or problems for individuals with low LR. (Rodriguez et al., Alcohol Clin Exp Res 17:155-161 (1993); Schuckit and Smith (1996); Volovka et al Arch Gen Psychiatry 53:258-263 (1996)). In addition, our group has reported a correlation for LR of 0.3 across two generations of subjects. Animal studies support the contention that genetic influences impact on LR (Crabbe et al. J Pharmacol Exp Ther 277:624-632 (1996)), and in some studies, rodents with low LR have been noted to consume higher amounts of alcohol. (Gill et al., Alcohol Clin Exp Res 21:106A (1997); Li et al., Behavior Genetics 23:163-170 (1993)).
As part of a larger study, 41 men, about 39 years old, were selected from among the first 113 completed 15-year follow-ups in a prospective study. The genotyping was performed selectively from the first group of consecutively ascertained sons of alcoholics and controls from among what will eventually be a larger population as part of an ongoing study. Seventeen subjects whose low level of response to ethanol (LR) at age 20 were in the lower third were compared on five polymorphisms of four genes with 24 men whose reactions to alcohol had been above the median. Fourteen men with the LL genotype of the serotonin transporter (5-HTT) polymorphism and seven men with the Ser385 genotype of the GABAA (xcex16) polymorphism were found to have demonstrated lower LR scores at about age 20 and were significantly at greater risk of alcoholism than the other genotypes for those loci. All four subjects with combined LL and Ser385 genotypes had developed alcoholism and demonstrated the lowest LR scores overall. There was no evidence that two polymorphisms of the 5-HT2A receptor gene and one of the 5-HT2C receptor gene were related to LR or alcoholism in this sample.
For this pilot study, blood samples were taken from the first 41 appropriate consecutive men among the first 113 who completed 15-year follow-ups in an ongoing study. In order to identify subjects who were clearly low on LR, the men were selected to represent the two poles of LR. These included the first 17 subjects who upon original alcohol challenge at about age 20 demonstrated an overall LR in the lowest third, and the 24 whose intensities of reaction to alcohol had been above the median (High LR). The alcohol challenges, evaluated changes from baseline for subjective feeling of intoxication, standing steadiness, and hormones after drinking 0.75 ml/kg of ethanol. (Schuckit and Gold, Arch Gen Psychiatry 45:211-216 (1986). The overall LR was evaluated as the change from baseline (pre-alcohol) to the time of peak blood alcohol concentration (BAC) (60 minute values) following alcohol consumption.
Previously, about ten years after initial testing, all 453 original subjects were located, and 450 (99.3%) were successfully evaluated (Schuckit and Smith 1996). The current pilot data were gathered as part of the ongoing 15-year follow-up of the same sample. For genotyping, blood was drawn at the time of the 15-year interview, and analyses performed blind to the phenotypic classification of the subjects. Five polymorphisms were genotyped from genomic DNA prepared from lymphoblastoid cell lines as follows. For 5HT2a T102C and His452Tyr, using methods described elsewhere, polymorphisms were typed by PCR and restriction enzyme assays (Ozaki et al., Biol Psychiatry 40:1267-1272 (1996)). 5HT2c Cys23Ser was genotyped by creating an artificial restriction site using a PCR primer that introduces a base substitution close to the codon of interest (Haliassos et al., Nucleic Acids Res. 17:3606 (1989)), and for 5HTTLPR, DNA amplification was accomplished using the two flanking primers used by Heils et al. (J. Neurochem 66:2621-2624 (1996)). This set of primers amplifies a 484 or 528 bp fragment corresponding to the 5HTTLPR short or long allele. The Ser385 polymorphism was genotyped using primers GABAaxcex16-9f and GABAaxcex16-9b: 5xe2x80x2CTG ACT CCA AAT ATC ATC TG3xe2x80x2 (SEQ. ID. NO. 9) and 5xe2x80x2GAG AAG CAT CTA CAC AAG TC3xe2x80x2 (SEQ. ID. NO. 10). Amplification with this set of primers resulted in a 367 bp PCR product containing a Fok I restriction site.
Genotypes for each of the five polymorphisms were evaluated against the three dependent variables provided in Table 2, with diagnoses based on the criteria of the Third Revised Diagnostic and Statistical Manual (DSM-III-R) of the American Psychiatric Association (1987). The differences across the two or three genotypes available at each locus were evaluated either by ANOVA or Student""s t-test for continuous variables, and chi-square (X2) with Yates"" correction for categorical data.
The 41 males selected for low and high LR were similar in age (about 39 years), marital status (about 73% married), and education (only 5% lacked a college education). Consistent with prior reports (Schuckit and Smith 1996), the low LR subjects were significantly more likely to have been diagnosed as alcohol dependent during the 15-year follow-up (64.7% vs. 8.3%, "khgr"2=14.6, df=1, p less than 0.002). There were no significant differences in the self-reported ethnic group of origin ("khgr"2=7.16, df=5, p=0.21), and all subjects were Caucasian.
Table 2 presents the genotype by trait analyses for each candidate gene locus. For the 5-HTT polymorphism, significantly lower LR. scores were observed at age 20 for the 14 subjects with the LL genotype (p=0.04). Consistent with these observations, subjects with the LL genotype were significantly more likely to have fulfilled criteria for alcohol abuse or dependence at some time during the 15 years of follow-up (p=0.04), but there were no differences across groups in the proportion with alcoholic relatives. Because of evidence that the 5-HTT S allele might act in a dominant fashion (Heils et al J Neurochem 66:2621-2624 (1996)), the combined SL and SS genotype groups were compared to the LL group, with results supporting an enhanced and statistically significant difference on the mean LR (xe2x88x920.89xc2x11.00 vs 0.16xc2x10.75, t=2.64, df=39, p=0.01), and on the proportion with an alcoholic diagnosis (57.1% vs 18.5%, X2=6.35, df=1, p=0.02). While not shown in the Table due to overlap with the mean LR score, we have also evaluated the proportion of subjects whose LR fell into the lowest third for comparability to prior publications. This proportion was also significantly higher for the LL group (71.4% LL, 31.6% SL, and 12.5% SS; X2=8.7, df=2, p=0.02.
Associations of the GABAAxcex16 genotype to both LR and alcoholism were also observed. Here, Pro/Ser heterozygous individuals were more likely than Pro385 homozygotes to be alcoholic (p=0.02), and they also demonstrated fronds for a lower LR score (p=0.06) and a higher proportion of FHPs (p=0.07). While not shown in Table 2, those with Ser385 were more likely to be in the lowest third in response to alcohol (71.4% vs. 35.3%, X2=3.12, df=1, p=0.08). Comparisons across the genotypes for the 5-HTT102C, 5-HT2Atyr His452tyr, and the 5-HT2c Cys23Ser polymorphisms demonstrated no relationship of genotypes to either LR scores or alcoholism diagnoses.
An analysis was also carried out combining information regarding the 5-HTT and GABAAxcex16 polymorphisms. The four men with a combination of both LL 5-HTT and Ser385 GABAA xcex16 genotypes (the two associated most closely with low LR and alcoholism) had the lowest LR at age 20 (xe2x88x921.29xc2x10.53 vs. xe2x88x920.74xc2x11.13 for LL/PP, 0.59xc2x11.11 for SL/PS, xe2x88x920.13xc2x10.88 for SL/PP and 0.03xc2x10.45 for SS/PPxe2x80x94test for linear trend t=2.86, df=4, p less than 0.007). All four of these subjects fell into the lowest third of LR, all were alcoholic, and all were FHP. At the other extreme, the eight men carrying both the SS 5-HTT and the Pro385 GABAA receptor genotypes (the two genotypes associated with the highest LR scores and nonalcoholic status) had the highest overall LR score, and were the least likely to be alcoholic. There was no evidence of an interaction between the 5-HTT and GABAAxcex16 genotypes (F(1,36)=0.01, p=0.92). As was true in the single locus analysis, these two-locus results reach even higher levels of statistical significance if all subjects with at least one copy of the dominant S allele are combined into one group.
The analysis above demonstrates the significant relationship to LR and alcoholism at two loci, the 5-HTTLPR serotonin transporter variant and the GABAA xcex16 amino acid substitution Ser385. Evidence supporting the importance of the GABAA receptor system in the effects of alcohol and the development of alcoholism is scant. The results above verify that the Pro385Ser polymorphism is indicative of LR and future alcoholism. Furthermore, the disclosure above clearly indicates that genotyping of the human xcex16 subunit of the GABAA receptor gene is useful for predicting whether an individual will exhibit a particular sensitivity to ethanol and/or benzodiazepine drugs.
The Existence of More Polymorphisms Associated with BZD and Ethanol Insensitivity
Those having ordinary skill in the art will appreciate that direct gene analysis using the SSCP technique may not be sufficiently sensitive to detect all possible sequence variants of the GABAA xcex16 receptor gene. In view of the fact that 40% of the GABAA xcex16 receptor gene coding sequence was not screened in this study, it is possible that unknown variants in either the coding sequence or promoter region of the xcex16 gene are in linkage disequilibrium with the Pro385Ser and G1031C variants described herein. In light of the findings presented above and the disclosure which follows, the discovery of more GABAA xcex16 receptor polymorphisms that are associated with ethanol and Benzodiazepine drug sensitivty would be routine. Regardless of the particular polymorphism used as a marker of genetically determined sensitivity to ethanol and benzodiazepine drugs, our findings indicate that methods based on xcex16 genotyping in humans can determine a predisposition to benzodiazepine/ethanol sensitivity. The section below describes many approaches that can be used to identify more polymorphisms in the GABAA xcex16 receptor gene that lead to BZD and ethanol insensitivity.
Characterization of Pro385Ser by Computational Analysis
Preliminary computational analysis revealed that the Pro385Ser polymorphism occurs in the second intracellular domain of the receptor near a putative protein kinase C phosphorylation site. Homology searches of nucleic acid and protein databases using software known to those of skill in the art can reveal other transmembrane proteins with a similar motif and can provide a greater understanding of how the Pro385Ser polymorphism confers BZD/ethanol insensitivity. Furthermore, by employing conventional approaches in protein modeling and methods of rational drug design, models of the three-dimensional structure of Pro385 or Ser385 GABAA xcex16 can be obtained and agents that interact or circumvent Pro385 or Ser385 GABAA xcex16 can be identified. Examples 3-6 disclose several software and hardware embodiments and provide computational methods that can be used to further characterize the Pro385 or Ser385 GABAA xcex16 nucleic acid and polypeptide sequences and develop drugs that interact with the Pro385 or Ser385 GABAA xcex16 receptors.
Aspects of the invention also include recombinant vectors, probes, and primers comprising Pro385 or Ser385 GABAA xcex16 sequence. The discussion below describes embodiments having Pro385 or Ser385 GABAA xcex16 nucleic acids.
Use of Nucleic Acids Encoding Pro385 or Ser385 GABAA xcex16, or Portions Thereof
The sequence of the Pro385 and Ser385 GABAA xcex16 is provided in the sequence listing (SEQ. ID NOs. 11 and 12). Wild-type and/or mutant Pro385 or Ser385 GABAA xcex16 sequences, their functional equivalents, or fragments of these sequences at least six nucleotides in length encoding Pro385 or Ser385 GABAA xcex16 can be used in accordance with embodiments of the invention. Preferably, the nucleic acid embodiments comprise at least 12, 15, or 17 consecutive nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom). More preferably, the nucleic acid embodiments comprise at least 20-30 consecutive nucleotides from the extended cDNA (or genomic DNAs obtainable therefrom). In some cases, the nucleic acid embodiments comprise more than 30 nucleotides from the nucleic acids encoding Pro385 or Ser385 GABAA xcex16, or portions thereof. In other cases, the nucleic acid embodiments comprise at least 40, at least 50, at least 75, at least 100, at least 150, or at least 200 consecutive nucleotides from the nucleic acids encoding Pro385 or Ser385 GABAA xcex16, or portions thereof. The nucleic acids listed in SEQ. ID Nos. 9-10 are desirable embodiments, for example. Subsequences comprising hybridizable portions of Pro385 or Ser385 GABAA xcex16 sequence have use, e.g., in nucleotide acid hybridization assays, Southern and Northern Blot analysis, etc., as will be described infra.
Some embodiments comprise recombinant nucleic acids having all or part of the Pro385 or Ser385 GABAA xcex16 gene. A recombinant construct can be capable of replicating autonomously in a host cell. Alternatively, the recombinant construct can become integrated into the chromosomal DNA of the host cell. Such a recombinant polynucleotide comprises a polynucleotide of genomic or cDNA, of semi-synthetic or synthetic origin by virtue of human manipulation. Therefore, recombinant nucleic acids comprising sequences otherwise not naturally occurring are provided by embodiments of this invention. Although Pro385 or Ser385 GABAA xcex16 as it appears in nature can be employed, it will often be altered, e.g., by deletion, substitution, or insertion and will be accompanied by sequence not present in a human.
The nucleotide acid sequence depicted in the sequence listing (SEQ. ID NO. 12) can be altered by mutation such as substitutions, additions, or deletions that provide for sequences encoding functionally equivalent molecules. According to one embodiment, a molecule is functionally equivalent or active compared with a molecule having the sequence depicted in SEQ. ID NO: 11 or 12 if it has the ability to confer ethanol or BZD insensitivity. Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as derived from the sequence listing (SEQ. ID NO: 11 or 12) can be used in some embodiments of the present invention. These include, but are not limited to, nucleic acid sequences comprising all or portions of the Pro385 or Ser385 GABAA xcex16 gene which have been altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
In addition, recombinant Pro385 or Ser385 GABAA xcex16-encoding nucleic acid sequences of the invention can be engineered so as to modify processing or expression of Pro385 or Ser385 GABAA xcex16. For example, and not by way of limitation, the Pro385 or Ser385 GABAA xcex16 gene can be combined with a promoter sequence and/or ribosome binding site, or a signal sequence can be inserted upstream of Pro385 or Ser385 GABAA xcex16 encoding sequences to permit secretion of Pro385 or Ser385 GABAA xcex16 and thereby facilitate harvesting or bioavailability. Additionally, a given Pro385 or Ser385 GABAA xcex16 nucleic acid can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction sites or destroy preexisting ones, or to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson et al., J. Biol. Chem. 253:6551 (1978)).
By using the Pro385 or Ser385 GABAA xcex16 nucleic acid sequences disclosed in the sequence listing (SEQ ID NO. 12), probes can be designed and manufactured by oligonucleotide synthesis and cDNA or genomic libraries can be screened so as to isolate natural sources of the nucleic acid embodiments and homologs thereof. Alternatively, such nucleic acids can be provided by amplification of sequences resident in genomic DNA or other natural sources by PCR. Example 7 describes the preparation of PCR primers and the amplification of Pro385 or Ser385 GABAA xcex16 DNA.
The nucleic acids of the invention can also be used as reagents in isolation procedures and diagnostic assays. For example, sequences from nucleic acids encoding Pro385 or Ser385 GABAA xcex16, or portions thereof can be detectably labeled and used as probes to isolate other sequences capable of hybridizing to them. In addition, sequences from nucleic acids encoding Pro385 or Ser385 GABAA xcex16, or portions thereof can be used to make PCR primers by conventional oligonucleotide synthesis for use in isolation and diagnostic procedures. The discussion that follows describes some of the expression constructs and protein embodiments of the invention.
Pro385 or Ser385 GABAA xcex16 Peptides and Their Expression
Pro385 or Ser385 GABAA xcex16 proteins, or fragments, or derivatives thereof include, but are not limited to, those containing as a primary amino acid sequence all or part of the amino acid sequence substantially as deduced from the sequence listed in SEQ. ID NO: 11 or 12 including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. Accordingly, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs. For example, the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In other aspects of the invention, Pro385 or Ser385 GABAA xcex16 proteins or fragments or derivatives thereof, which are differentially modified during or after translation, e.g., by phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, linkage to an antibody molecule, membrane molecule, or other ligand, are contemplated. (Ferguson et al., Ann. Rev. Biochem. 57:285-320 (1988)).
In one embodiment, the inventors contemplate Pro385 or Ser385 GABAA xcex16 or a portion thereof, in a cell line. Further, the present inventors envision isolating or purifying Pro385 or Ser385 GABAA xcex16 protein. Example 8 provides several approaches to synthesize, express, and isolate or purify the Pro385 or Ser385 GABAA xcex16 protein, or fragments thereof. The term xe2x80x9cisolatedxe2x80x9d requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or protein present in a living cell is not isolated, but the same nucleic acid or protein, separated from some or all of the coexisting materials in the natural system, is isolated. In accordance with this definition, Pro385 or Ser385 GABAA xcex16 nucleic acid or protein or Pro385 or Ser385 GABAA xcex16 nucleic acid or polypeptide fragments present in a cell lysate are xe2x80x9cisolatedxe2x80x9d. The term xe2x80x9cpurifiedxe2x80x9d does not require absolute purity; rather it is intended as a relative definition. For example, recombinant nucleic acids and proteins are routinely purified to electrophoretic homogeneity, as detected by ethidum bromide staining or Coomassie staining, and are suitable in several assays despite having the presence of contaminants. Example 8 provides several approaches to synthesize, express, and isolate or purify the Pro385 or Ser385 GABAA xcex16 protein, or fragments thereof. Following synthesis or expression and purification of the proteins encoded by the Pro385 or Ser385 GABAA xcex16 nucleic acid or portion therof, the purified proteins can be used to generate antibodies as described in the following section.
Production of an Antibody to a Pro385 or Ser385 GABAA xcex16 Polypeptide
The antibodies contemplated have many uses including, but not limited to, biotechnological applications, therapeutic/prophylactic applications, and diagnostic applications. While antibodies capable of specifically recognizing the protein of interest can be generated using synthetic 15-mer peptides having a sequence encoded by Pro385 or Ser385 GABAA xcex16 gene or portion thereof by injecting the synthetic peptides into mice to generate antibody, a more diverse set of antibodies can be generated using recombinant or purified Pro385 or Ser385 GABAA xcex16 protein or fragments thereof, as described in Example 9. The discussion that follows describes several diagnostic embodiments of the invention.
Diagnostic Embodiments
Generally, the diagnostics and methods of use thereof can be classified according to whether the diagnostic detects the presence of Pro385 or Ser385 GABAA xcex16 nucleic acid in a sample or Pro385 or Ser385 GABAA xcex16 protein in a sample. Accordingly, the detection of the Pro385 or Ser385 GABAA xcex16 nucleic acid and/or protein in a biological sample indicates a predilection to BZD/ethanol insensitivity. Additionally, the manufacture of kits which incorporate the reagents and methods described in the following embodiments so as to allow for the rapid detection of the Pro385Ser polymorphism are contemplated. The diagnostic kits can include a nucleic acid probe or an antibody that specifically detects the Pro385Ser polymorphism, for example, or can detect both wild-type and mutant. The detection component will typically be supplied in combination with one or more of the following reagents. A substratum capable of absorbing or otherwise binding DNA, RNA, or protein will often be supplied. Available substrata for this purpose includes membranes of nitrocellulose, nylon or derivatized nylon that can be characterized by bearing an array of positively charged substituents. One or more restriction enzymes, such as FokI, can be furnished in the kit, as can non-human polynucleotides like calf-thymus or salmon-sperm DNA.
Useful nucleic acid-based diagnostic techniques include, but are not limited to, fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern Blot analysis, single-stranded confirmation analysis (SSCA), RNase protection assay, dot blot analysis, and PCR. The starting point for these analysis is isolated or purified DNA from a biological sample. Most simply, blood is drawn from the subject to be tested and DNA extracted from the nucleated cells in the blood. In some cases, primers corresponding to regions of Pro385Ser can be used with PCR to amplify this DNA so that it can be more easily be detected in diagnostic applications.
Several methods can be used to detect the Pro385Ser polymorphism in a biological sample. Direct DNA sequencing, either manual sequencing or automated fluorescent sequencing can detect sequence variation. Another approach is the single-stranded confirmation polymorphism assay (SSCA) (Orita et al., Proc. Natl. Acad. Sci. USA 86:2776-2770 (1989)) discussed above. This method, however, does not detect all sequence changes, especially if the DNA fragment size is greater than 200 base pairs, but can be optimized to detect most DNA sequence variation. The reduced detection sensitivity is a disadvantage, but the increased throughput possible with SSCA makes it an attractive, viable alternative to direct sequencing for mutation detection. The fragments which have shifted mobility on SSCA gels are then sequenced to determine the exact nature of the DNA sequence variation. Other approaches based on the detection of mismatches between the two complimentary DNA strands include clamped denaturing gel electrophoresis (CDGE) (Sheffield et al., Am. J. Hum. Genet. 49:699-706 (1991)), heteroduplex analysis (HA) (White et al., Genomics 12:301-306 (1992)), and chemical mismatch cleavage (CMC) (Grompe et al., Proc. Natl. Acad. Sci. USA 86:5855-5892 (1989)). A review of currently available methods of detecting DNA sequence variation can be found in a recent review by Grompe, Nature Genetics 5:111-117 (1993).
A rapid preliminary analysis to detect polymorphisms and DNA sequences can be performed by looking at a series of Southern Blots of DNA cut with one or more restriction enzymes preferably with a large number of restriction enzymes. Each block contains lanes of DNA from uninfected individuals and the DNA to be tested. Southern Blots displaying hybridizing fragments when probed with sequences corresponding to Pro385Ser indicate the presence of the genotype associated with BZD/ethanol insensitivity. Detection of point mutations can also be accomplished by amplifying the DNA directly from the sample using primers corresponding to the regions of Pro385Ser by standard PCR techniques. The DNA sequence of the amplified sequences can then be determined.
There are six well-known methods for confirming the presence of Pro385Ser: 1) single-stranded confirmation analysis (SSCA) (Orita et al.); 2) denaturing gradient gel electrophoresis (DGGE) (Wartell et al., Nucl. Acids Res. 18:2699-2705 (1990), Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236 (1989), 3) RNase protection assays (Finkelstein et al., Genomics 7:167-172 (1990), Kinszler et al., Science 251:1366-1370 (1991)); 4) the use of proteins which recognize nucleotide mismatches, such as the E. Coli mutS protein (Modrich, Ann. Rev. Genet. 25:229-253 (1991); and allele-specific PCR (Rano and Kidd, Nucl. Acids Res. 17:8392 (1989)). For allele-specific PCR, primers are used which hybridize at their 3xe2x80x2 ends to the Ser385 GABAA xcex16 mutation. If the particular Ser385 mutation is not present, an amplification product is not observed. Amplification Refractory Mutation System (ARMS) can also be used, as disclosed in European Patent Application Publication No. 0332435 and in Newton et al., Nucl. Acids Res. 17:2503-2516 (1989).
In the first methods (SSCA, DGGE, and RNase protection assay), a new electrophoretic band appears. SSCA detects a band which migrates differentially because the sequence change causes a difference in single-strand, intramolecular base pairing. RNase protection involves cleavage of the mutant polynucleotide into two or more smaller fragments. DGGE detects differences in migration rates of sequences compared to wild-type sequences, using a denaturing gradient gel. In an allele-specific oligonucleotide assay (ASOs) (Conner et al., Proc. Natl. Acad. Sci. USA 80:278-282 (1983)), oligonucleotide is designed which detects a specific sequence, and an assay is performed by detecting the presence or absence of hybridization signal. In the mutS assay, the protein binds only to sequences that contain a nucleotide mismatch in a heteroduplex between mutant and wild-type sequences.
Mismatches, according the present invention, are hybridized nucleic acid duplexes in which the two strands are not 100% complementary. Lack of total homology can be due to deletions, insertions, inversions, or substitutions. Mismatched detection can be used to detect point mutations in the gene or in its mRNA product. While these techniques are less sensitive than sequencing, they are simpler to perform on a large number of biological samples.
An example of a mismatch cleavage technique is the RNase protection method. In practice, the method involves the use of a labeled riboprobe which is complementary to the Ser385 gene coding sequence. The riboprobe and either mRNA or DNA isolated from the biological sample are annealed (hybridized) together and subsequently digested with the enzyme RNase A which is able to detect some mismatches in a duplex RNase structure. If a mismatch is detected by RNase A, it cleaves at the site of the mismatch. Thus, when the annealed RNA is separated on a electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNase A, an RNA product will be seen which is much smaller than the full length duplex RNA for the riboprobe and the mRNA or DNA. The riboprobe need not be the fall length of Pro385 or Ser385 GABAA xcex16 mRNA or gene, it will be desirable to use a number of these probes to screen the whole mRNA sequence for mismatches. In a similar fashion, DNA probes can be used to detect mismatches, through enzymatic or chemical cleavage. See, e.g., Cotton, et al., Proc. Natl. Acad. Sci. USA 85:4397 (1988), Shenk et al., Proc. Natl. Acad. Sci. USA 72:989 (1975), Novack et al., Proc. Natl. Acad. Sci. USA 83:586 (1986).
Alternatively, mismatches can be detected by shifts in the electrophoretic ability of mismatched duplexes relative to matched duplexes. (See, e.g., Cariello, Human Genetics 42:726 (1988)). With either riboprobes or DNA probes, the cellular mRNA or DNA which might contain the Ser385 gene can be amplified by PCR before hybridization. DNA sequences isolated from biological samples which have been amplified by use of PCR can be screened using allele-specific probes. These probes are nucleic acid oligomers, each of which contains a region of Ser385 sequence. For example, one oligomer can be about 30 nucleotides in length and corresponds to a portion of the Ser385 sequence referenced in SEQ ID NO. 12 By use of a battery of such allele-specific probes, PCR amplification products can be screened to identify the presence of the Pro385Ser polymorphism. Hybridization of allele-specific probes with amplified Pro385 or Ser385 GABAA xcex16 sequences can be performed, for example, on a nylon filter.
The most definitive test for the presence of the Pro385Ser polymorphism, is to directly compare nucleotide or protein sequences isolated from a biological sample with those from a control population. The control population, for example, can contain DNA, RNA, cDNA nucleic acids samples representative of the wild-type sequence, as listed in SEQ ID NO. 11 (experimental being the Pro385Ser polymorphism, as listed in SEQ ID NO. 12) Alternatively, one could sequence messenger RNA after amplification, e.g., by PCR. Examples 10-13 describe nucleic acid-based diagnostic methods of the invention.
In addition to the diagnostics and methods based on the detection of nucleic acid that encodes to Pro385 or Ser385 GABAA xcex16, diagnostics and methods based on the detection of Pro385 or Ser385 GABAA xcex16 proteins are also envisioned. The following discussion details several embodiments of this aspect of the invention.
Protein-Based Diagnostic Embodiments
The presence of Pro385 or Ser385 GABAA xcex16 protein can be detected by screening for the presence of the protein using conventional assays. For example, monoclonal antibodies immunoreactive with Pro385 GABAA xcex16 protein can be used to screen biological samples for the presence of the Pro385Ser polymorphism. Similarly, antibodies specific for or Ser385 GABAA xcex16 protein can be used to screen for the presence of the Pro385Ser polymorphism. Such immunological assays can be done in many convenient formats.
In a preferred embodiment, antibodies will immunoprecipitate specifically the Ser385 GABAA xcex16 protein (e.g., will not cross-react with the Pro385 protein) from solution as well as react specifically with Ser385 protein on Western or Immunoblots of polyacrylamide gel. In another preferred embodiment, antibodies will detect Ser385 in paraffin or frozen sections, using immunocytochemical techniques. Preferred embodiments relating to methods for detecting the Pro385 or Ser385 GABAA xcex16 protein include enzyme-linked immunosorbant assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies. Exemplary sandwich assays are described by David et al., in U.S. Pat. Nos. 4,376,110 and 4,486,530, hereby incorporated by reference.
We also contemplate the preparation of diagnostic kits comprising antibodies specific for the Ser385 protein and not cross-reactive with the Pro385 protein. Such kits can also include nitrocellulose or nylon membranes for immobilizing protein from a biological sample to be tested. Results from the kit assays can be interpreted by a healthcare provider or a diagnostic laboratory. Alternatively, diagnostic kits are manufactured and sold to private individuals for self-diagnosis. In addition, we contemplate the design and manufacture of supports having Pro385 or Ser385 GABAA xcex16 polypeptides, as described below, for use in methods for identifying agents that interact with the Pro385 or Ser385 GABAA xcex16 polypeptides.
Construction of a Multimeric Support Having Pro385 or Ser385 GABAA xcex16 or Polypeptide Fragments Thereof
A biotechnological tool that is useful for the discovery of agents that interact with Pro385 or Ser385 GABAA xcex16 or circumvent Pro385 or Ser385 GABAA xcex16 (e.g., high throughput screening) desirably provide Pro385 or Ser385 GABAA xcex16 in such a form or in such a way that a sufficient affinity for the agent is obtained. For example, while monomeric Pro385 or Ser385 GABAA xcex16 (i.e. appearing as a discrete unit of the protein on a support) is sufficiently active to mediate association with an agent, multimeric Pro385 or Ser385 GABAA xcex16 (i.e. appearing as multiple units of the protein on a support) can have a far greater affinity for the agent and would provide sufficient binding events to be detectable by conventional methods. Example 14 provides several approaches that can be used to create a multimeric support having Pro385 or Ser385 GABAA xcex16 or polypeptide fragments thereof. The multimeric supports can be used in methods of high throughput screening, several of which are described in the following section.
High Throughput Screening for Agents That Interact With Pro385 or Ser385 GABAA xcex16 Protein, or Polypeptide Fragments Thereof
High throughput screening is an approach to drug discovery that searches for a protein, peptide, peptidomimetic, or chemical that will interact on a defined target such as the Pro385 or Ser385 GABAA xcex16 protein, or a polypeptide fragment thereof. Generally, a library of proteins, polypeptides, peptides, peptidomimetics, or chemicals, collectively referred to as xe2x80x9cagentsxe2x80x9d, is screened against the target in biological assays and agents which interact with the target are identified and used directly as therapeutics or as a basis to develop new therapeutics using combinatorial chemistry and protein modeling. Example 15 provides several high throughput screening methods that are used to identify agents that interact with Pro385 or Ser385 GABAA xcex16 protein or polypeptide fragments thereof.
Example 1 describes the methods that were used to measure BZD sensitivity in human subjects.